Downstream Targets of Dopamine Receptor D3 Activation in the Mouse Pancreatic β-Cells

Abstract

We have shown that dopamine modulates a negative feedback control of insulin secretion from mouse islets by activating the dopamine receptor D3 (DRD3). Receptor activation causes a dose-dependent reduction of the frequency of glucose-triggered intracellular calcium oscillations, which correlates with the observed decrease of insulin secretion. Although it is known that DRD3 can inhibit adenylate cyclase (AC), activate potassium channels, and inhibit calcium channels, the downstream molecular targets of the DRD3 signal transduction cascade in the islet are still unknown. The goal of this study is to identify the main signaling pathway mediating the DRD3-driven inhibition of insulin secretion. Preliminary data suggests that DRD3 signaling in β-cells proceeds through modulation of ion channels rather than by inhibition of AC.We used patch clamp to study the effect of dopamine on the ion channels. Based on these experiments we selected molecular targets to investigate further. We have tagged fluorescent proteins on to the Gβγ complex of the heterotrimeric G protein, and also on to candidate-target ion channels. This allows us to use quantitative fluorescence analysis tools to measure the changes in protein-protein interaction before and after pharmacological stimulation of DRD3. Data is analyzed using a spatial intensity distribution analysis (Godin, A. G. et al., PNAS, 2011) to detect protein interactions in live cells and fixed samples. This analysis allow us to compare results from cell lines overexpressing our proteins of interest with the results from native pancreatic islets from mice and human donors where immuno-staining techniques can be used to label the proteins under investigation.The information gained will help us understand how pancreatic islets regulate insulin secretion. Moreover, this signaling pathway could potentially be exploited to design effectors of insulin secretion as a treatment for type-2 diabetes.